Immunochromatography detection of multidrug-resistant staphylococcus and diagnostic kit

ABSTRACT

It is intended to provide an immunochromatographic detection device which detects PBP2′ specifically produced by a multidrug-resistant  Staphylococcus  bacterium by an immunochromatographic detection method sensitively, simply and rapidly and is capable of determining infection with a multidrug-resistant  Staphylococcus , a diagnostic method using the detection device, and a diagnostic kit including the detection device. The invention is directed to the immunochromatographic detection device for detecting a multidrug-resistant  Staphylococcus  which includes a sample supply unit which supplies a sample solution which is assumed to contain a multidrug-resistant  Staphylococcus  or a solution which is assumed to contain PBP2′ released from a cell wall by a pretreatment of a sample onto a sheet-shaped solid-phase support, a labeling reagent unit which retains a labeling reagent in which an antibody specifically binding to PBP2′ is labeled spreadable on the solid-phase support, and a trapping reagent unit in which a trapping reagent capable of trapping a complex of PBP2′ and the labeling reagent by specifically binding to the complex is immobilized, and in which an amphoteric surfactant; an anionic surfactant and/or a nonionic surfactant is contained in the trapping reagent unit or in the solid-phase support upstream of the trapping reagent unit.

TECHNICAL FIELD

The present invention relates to immunochromatography detection and akit utilizing the same.

BACKGROUND ART

Staphylococci includes 40 or more bacterium types roughly classifiedinto Staphylococcus aureus and coagulase-negative staphylococci(hereafter abbreviated to “CNS”) from the clinical point of view.Staphylococcus aureus are regarded as pathogenic organisms and CNS areregarded as nonpathogenic organisms.

Infections are treated with the use of antibiotics. Since manystaphylococci are drug-resistant, they are classified based on drugresistance from the clinical point of view.

Among Staphylococcus aureus that are clinically important pathogenicorganisms, methicillin-resistant Staphylococcus aureus (MRSA) areStaphylococcus aureus that show resistance to β-lactam agents, includingpenicillin such as methicillin. Also, many such bacteria show resistanceto many drugs such as aminoglycosides and macrolides. Thus, suchbacteria are clinically regarded as multidrug-resistant Staphylococcusaureus. In contrast, Staphylococcus aureus showing sensitivity tomethicillin are referred to as methcillin-sensitive Staphylococcusaureus (hereafter referred to as “MSSA”).

Staphylococcus aureus produce a variety of toxins, includingenterotoxin, toxic-shock syndrome toxin, hemolysin, and exfoliativetoxin (Hideo IGARASHI: TSST-1, “Shinshu to meneki (Invasion andimmunity),” 3, 3-10, 1994). Infection with such toxins would causeenteritis, pneumonia, dermatitis, organic failure, or the like, andserious infection may lead to death. When Staphylococcus aureus areisolated from a patient, accordingly, whether or not the bacterium isMRSA must be inspected as quickly as possible. In the event of MRSAinfections, adequate drugs, such as vancomycin or arbekacin sulfate,which are regarded as being effective against MRSA, must be selected andadministered to a patient.

Staphylococci other than Staphylococcus aureus are indigenous bacteria,and they are nonpathogenic to healthy individuals in general. When anorgan transplant patient takes an immunosuppressant as a measure toprevent a postoperative infection or in the case of a so-calledcompromised patient with a weakened immune system due to anaging-induced weakened physical strength, however, opportunisticinfections may occur.

Some staphylococci other than Staphylococcus aureus have acquiredmethicillin resistance or multidrug resistance, and these bacteria arecollectively referred to as methicillin-resistant coagulase-negativestaphylococci (hereafter abbreviated as “MRCNS”) or multidrug-resistantcoagulase-negative staphylococci, respectively. As with the case ofMRSA, drugs that are effective against MRCNS are limited if acompromised patient is infected therewith. Thus, MRCNS has been amedical problem.

MRSA and MRCNS are collectively referred to as multidrug-resistantstaphylococcus.

The drug resistance of MRSA or MRCNS is known to result from expressionof a new enzyme, PBP2′, in addition to four types of penicillin-bindingproteins (i.e., PBP1, PBP2, PBP3, and PBP4) that crosslink mureinchains, which are constitutional elements of the cell wall ofstaphylococci and which synthesize the cell wall (Utsui, Y., and Yokota,T.: Role of an altered penicillin-binding protein in methicillin- andcephem-resistant Staphylococcus aureus. Antimicrobial Agents andChemotherapy, 28, 397-403, 1985). PBP1 to PBP4 proteins thatstaphylococci possess in common are inactivated ascell-wall-synthesizing enzymes by penicillin antibiotics, which aresubstrate analogues, and bacteria eventually die when synthesis of cellwalls becomes unfeasible. However, MRSA and MRCNS express a newcell-wall-synthesizing enzyme, PBP2′, which exhibits little affinity toβ-lactam antibiotic substances, i.e., which would not be inactivatedthereby. MRSA and MRCNS are considered to proliferate by altering rolesin cell wall synthesis. Most MRSA and MRCNS acquire mechanisms ofresistance to other antibiotics and become multidrug-resistant bacteriathat are resistant to many antibiotics. Such bacteria are regarded asmultidrug-resistant staphylococci instead of staphylococci havingresistance merely to β-lactam antibiotics.

General techniques for separating and identifying staphylococci involvethe use of nasal cavity swabs, pharyngeal swabs, sputum, blood, pus,stool, or other samples as clinical samples, and isolation culturethereof with the use of an agar medium or liquid agar medium isperformed. When cultured in an agar medium, colonies suspected of beingstaphylococci are selected from among the grown colonies and furthersubjected to pure culture, and staphylococci or Staphylococcus aureusare identified via microscopic visualization of Gram-stained images orbiochemical character tests of coagulase production capacity or mannitoldegradation capacity. When cultured in a liquid medium, a culturesolution is sowed on an agar medium and cultured therein for colonyisolation, and colonies suspected of being staphylococci are alsosubjected to pure culture followed by identification. The bacteria thathave been identified as the Staphylococcus aureus or staphylococci aresubjected to a drug-sensitivity test or the like, and whether or not abacterium of interest is MRSA, MSSA, or MRCNS is determined based ontest results. A drug-sensitivity test is generally carried out byculture, such as a dilution technique or a disk sensitivity test. Suchdrug sensitivity test is known to require a culture duration of 16 to 24hours (it would take 3 or more days from separation of clinical samplesto determination if both isolation culture and pure culture areconducted) and to produce differences in test results due to, forexample, the concentration of bacteria, culture temperature, mediumcomposition, or drug to be used. Thus, a person who conducts such testis required to be highly experienced with the procedure.

In recent years, a method that detects the mecA gene encoding PBP2′,which is a main body of a drug-resistant mechanism, via pure culture,isolation culture, or directly from a clinical sample via PCR toevaluate antibody resistance based on the conditions of the mecA genecarried in the analyte bacterium has been developed. However, the factthat a bacterium carries the mecA gene does not always mean theexpression of antibiotic resistance, and some bacteria have not acquiredresistance even though they carry such gene.

As described above, production of PBP2′ plays a key role in theexpression of multidrug resistance, and detection of PBP2′ fromstaphylococci can be a useful means for learning whether or not abacterium of interest has acquired resistance.

PBP2′ produced specifically by bacteria of the multidrug-resistantstaphylococcus, including MRSA, is detected by anantigen-antibody-reaction-based immunological means, such as Westernblotting, radioimmunoassay, or a slide latex agglutination test (JPPatent No. 3638731). Such methods of detecting PBP2′, however, sufferfrom the following problems. Western blotting is complicated in terms ofprocedure, and it is difficult to rapidly process many samples.Radioimmunoassay is not practical from the viewpoint of routine testing,since it involves the use of a radioisotope, it requires BF separationinvolving separating an antigen-antibody complex from other non-bindingantigens or antibodies during assay, and it requires several hours tocomplete assay due to the presence of a denaturing agent used forextracting an antigen from the bacterium in the reaction system. Theslide latex agglutination test requires detection from axenic bacteriadue to false-positive reactions caused by contaminating bacteria (e.g.,false-positive reactions due to nonspecific reactions) and lowsensitivity. Thus, culture must be conducted at least twice, i.e.,isolation culture and pure culture, which in turn requires 2 to 3 daysto complete the determination, it increases the cost for mediums forpure culture, and it may cause a false-positive reaction due toagglutination after the determination time, even though such time can beas short as 3 minutes. It is also laborious due to the need ofcentrifugation for separating a PBP2′-containing supernatant fromcell-wall-derived pieces at the time of pretreatment of extracting PBP2′from the bacterium, which may contaminate the environment of thelaboratory, it involves complicated procedures such as transfer of asupernatant after centrifugation via pipetting, and it involves boiling.Accordingly, a method that can rapidly detect PBP2′ producedspecifically by a bacterium of multidrug-resistant staphylococcus withhigh specificity and sensitivity has been awaited as a replacement forconventional detection techniques.

DISCLOSURE OF THE PRESENT INVENTION Object to be Attained by theInvention

The present invention is intended to provide an immunochromatographydetection device that can detect PBP2′ produced specifically by abacterium of multidrug-resistant staphylococcus with high sensitivity ina simple and rapid manner via immunochromatography detection todetermine infection with multidrug-resistant staphylococcus, adiagnostic method using such detection device, and a diagnostic kitcomprising such detection device.

Means for Attaining the Object

The present inventors have conducted concentrated studies regarding amethod for extracting PBP2′ from a bacterium of multidrug-resistantstaphylococcus and assaying the same in a simple and rapid mannerwithout complicated procedures. They succeeded in assaying PBP2′ in asimple manner with the use of an immunochromatography detection deviceusing a reagent that is a labeled antibody binding specifically to PBP2′and a capture reagent that can specifically bind to and capture acomplex of PBP2′ and the labeled reagent. Further, they discovered thatPBP2′ could be extracted and assayed without the need for complicatedcentrifugation or other means by treating a sample with an alkalinesolution before assay, neutralizing the same, and applying the same tothe immunochromatography detection device. Further, they discovered thatassay can be carried out without causing a false-positive reaction byapplying a surfactant such as an ampholytic surfactant to a capturereagent site of the immunochromatography detection device on which thecapture reagent has been immobilized. This has led to the completion ofthe present invention.

More specifically, the present invention is as follows.

[1] A method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, comprising detection of acell-wall-synthesizing enzyme, PBP2′, via immunochromatography detectionbased on an antigen-antibody reaction.

[2] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to [1], which involvesthe use of an immunochromatography detection device comprising on asheet-like solid-phase support: a sample supply site to which a samplesolution deduced to contain a bacterium producing acell-wall-synthesizing enzyme, PBP2′ or a solution deduced to containPBP2′ released from the cell wall via sample pretreatment is supplied; alabeled reagent site that holds a reagent, which is a labeled antibodybinding specifically to PBP2′, in a manner such that the reagent is ableto spread across the solid-phase support; and a capture reagent site onwhich a capture reagent capable of specifically binding to and capturinga complex of PBP2′ and the labeled reagent has been immobilized.

[3] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to [1] or [2], whereinPBP2′ comes into contact with the labeled reagent at a site separatedfrom a solid-phase support in advance, and a sample-reagent mixturecomprising a sample solution deduced to contain a bacterium producing acell-wall-synthesizing enzyme, PBP2′ or a solution deduced to containPBP2′ released from the cell wall via sample pretreatment and thelabeled reagent that is a labeled antibody binding specifically to PBP2′is supplied to the sample supply site.

[4] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to [2] or [3], whereinthe labeled reagent is an insoluble carrier to which an antibody isbound.

[5] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to any one of [2] to[4], wherein the capture reagent site comprises an ampholyticsurfactant, an anionic surfactant, and/or a nonionic surfactant.

[6] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to any one of [2] to[5], wherein the capture reagent site comprises a sulfobetaine-typesurfactant.

[7] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to [5] or [6], whereinan anion having a high ionization tendency is added to a sample solutiondeduced to contain a bacterium producing a cell-wall-synthesizingenzyme, PBP2′, or a solution deduced to contain PBP2′ released from thecell wall via sample pretreatment prior to supply of such solution to asample supply site.

[8] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to [7], wherein theanion having a high ionization tendency is at least one anion selectedfrom the group consisting of a chloride ion, a bromide ion, and aniodide ion.

[9] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to any one of [5] to[8], wherein a cation having a high ionization tendency is added to asample solution deduced to contain a bacterium producing acell-wall-synthesizing enzyme, PBP2′, or a solution deduced to containPBP2′ released from the cell wall via sample pretreatment prior tosupply of such solution to a sample supply site.

[10] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to [9], wherein thecation having a high ionization tendency is at least one cation selectedfrom the group consisting of a potassium ion, a calcium ion, a sodiumion, and a magnesium ion.

[11] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to any one of [1] to[11], which comprises a step of pretreating a sample via alkalinetreatment or neutralization.

[12] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to [11], wherein thealkaline treatment is carried out using an aqueous solution of alkalimetal hydroxide or carbonate or an aqueous solution of alkaline earthmetal hydroxide or carbonate.

[13] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to [12], wherein the pHof the aqueous solution of alkali metal hydroxide or carbonate or theaqueous solution of alkaline earth metal hydroxide or carbonate is 11 orhigher.

[14] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to [12] or [13], whereinthe concentration of the alkali metal hydroxide or carbonate or alkalineearth metal hydroxide or carbonate is between 0.01N and 1.0N.

[15] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to any one of [11] to[14], wherein the aqueous solution after alkali treatment is neutralizedwith a buffer.

[16] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to any one of [1] to[15], wherein the sample supply site comprises glass fibers.

[17] The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to any one of [1] to[16], wherein the bacterium producing a cell-wall-synthesizing enzyme,PBP2′, is a multidrug-resistant staphylococcus.

[18] An immunochromatography detection device for detecting a bacteriumproducing a cell wall synthesizing enzyme, PBP2′, comprising on asheet-like solid-phase support: a sample supply site to which a samplesolution deduced to contain a bacterium producing acell-wall-synthesizing enzyme, PBP2′ or a solution deduced to containPBP2′ released from the cell wall via sample pretreatment is supplied; alabeled reagent site that holds a reagent, which is a labeled antibodybinding specifically to PBP2′, in a manner such that the reagent is ableto spread across the solid-phase support; and a capture reagent site onwhich a capture reagent capable of specifically binding to and capturinga complex of PBP2′ and the labeled reagent has been immobilized, thecapture reagent site comprising an ampholytic surfactant, an anionicsurfactant, and/or a nonionic surfactant.

[19] The immunochromatography detection device for detecting a bacteriumproducing a cell wall synthesizing enzyme, PBP2′, according to [18],wherein the capture reagent site comprises a sulfobetaine-typesurfactant.

[20] The immunochromatography detection device for detecting a bacteriumproducing a cell wall synthesizing enzyme, PBP2′, according to [18] or[19], wherein the sample supply site comprises glass fibers.

[21] The immunochromatography detection device for detecting a bacteriumproducing a cell wall synthesizing enzyme, PBP2′, according to any oneof [18] to [20], wherein the bacterium producing acell-wall-synthesizing enzyme, PBP2′, is a multidrug-resistantstaphylococcus.

[22] A kit for detecting a bacterium producing a cell-wall-synthesizingenzyme, PBP2′, comprising: the immunochromatography detection device fordetecting a bacterium producing a cell wall synthesizing enzyme, PBP2′,according to any one of [18] to [21]; and an anion or cation solutionhaving a high ionization tendency to be added to a sample solutiondeduced to contain a bacterium producing a cell-wall-synthesizingenzyme, PBP2′, or a solution deduced to contain PBP2′ released from thecell wall via sample pretreatment.

[23] The kit for detecting a bacterium producing acell-wall-synthesizing enzyme, PBP2′, according to [22], wherein theanion having a high ionization tendency is at least one anion selectedfrom the group consisting of a chloride ion, a bromide ion, and aniodide ion.

[24] The kit for detecting a bacterium producing acell-wall-synthesizing enzyme, PBP2′, according to [22], wherein thecation having a high ionization tendency is at least one cation selectedfrom the group consisting of a potassium ion, a calcium ion, a sodiumion, and a magnesium ion.

[25] The kit for detecting a bacterium producing acell-wall-synthesizing enzyme, PBP2′, according to any one of [22] to[24], which further comprises an alkali solution for alkali treatment ofa sample with alkali and a buffer for neutralization.

[26] The kit for detecting a bacterium producing acell-wall-synthesizing enzyme, PBP2′, according to [25], wherein thealkali solution is an aqueous solution of alkali metal hydroxide orcarbonate or an aqueous solution of alkaline earth metal hydroxide orcarbonate.

[27] The kit for detecting a bacterium producing acell-wall-synthesizing enzyme, PBP2′, according to [26], wherein the pHof the aqueous solution of alkali metal hydroxide or carbonate or theaqueous solution of alkaline earth metal hydroxide or carbonate is 11 orhigher.

[28] The kit for detecting a bacterium producing acell-wall-synthesizing enzyme, PBP2′, according to [26] or [27], whereinthe concentration of the alkali metal hydroxide or carbonate or alkalineearth metal hydroxide or carbonate is between 0.01N and 1.0N.

[29] The kit for detecting a bacterium producing acell-wall-synthesizing enzyme, PBP2′, according to any one of [22] to[28], wherein the bacterium producing a cell-wall-synthesizing enzyme,PBP2′, is a multidrug-resistant staphylococcus.

EFFECTS OF THE INVENTION

Use of the immunochromatography detection device of the presentinvention enables detection of PBP2′ with high sensitivity. This thenenables testing with one colony after isolation culture. Consequently,whether or not a bacterium of interest is MRSA or MRCNS can be evaluatedvia a single operation of culture in 1 day after separation thereof fromthe clinical sample. This can remarkably reduce testing duration and thecost of mediums used for pure culture. Further, blood (liquid) culturethat is carried out for testing MRSA bacteremia or septicemia enablesdirect detection from a positive medium. This enables initiation ofeffective treatment at an early stage, which leads to reduction oftreatment duration.

Also, centrifugation becomes unnecessary by imparting filtration effectsto a sample application member on a support on which an antibody hasbeen immobilized. Thus, sanitary laboratory conditions can bemaintained.

A capture site of the immunochromatography detection device may beimpregnated with a surfactant, so that binding of fractured bacteriaother than antigens to the labeled reagent and/or the capture site canbe prevented; i.e., false-positive reactions can be prevented, andsensitivity can be improved. This makes a previously necessary boilingprocedure in the process of bacterium pretreatment unnecessary.

This description includes part or all of the contents as disclosed inthe description and/or drawings of Japanese Patent Application No.2005-360984, which is a priority document of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment of the detection device of the presentinvention comprising a labeled reagent site.

FIG. 2 shows an embodiment of the detection device of the presentinvention comprising no labeled reagent site.

DESCRIPTION OF REFERENCE NUMERALS

-   1: A sample supply site-   2: A labeled reagent site-   3: A capture reagent (capture antibody) site-   4: A control site-   5: A solid-phase support (nitrocellulose membrane)-   6: An absorption site (absorbent pad)-   7: A top laminate or housing

BEST MODES FOR CARRYING OUT THE INVENTION

Hereafter, the present invention is described in detail.

The present invention relates to an immunochromatography detectiondevice that can detect PBP2′ that is present specifically in a bacteriumproducing a cell-wall-synthesizing enzyme, PBP2′, such as amultidrug-resistant staphylococcus, with high sensitivity in a simpleand rapid manner via immunochromatography detection to determineinfection with a bacterium producing a cell-wall-synthesizing enzyme,PBP2′, such as a multidrug-resistant staphylococcus, and a diagnostickit. The term “sample” used herein refers to a solution deduced tocontain a bacterium producing a cell-wall-synthesizing enzyme, PBP2′,and more specifically, a solution deduced to contain amultidrug-resistant staphylococcus bacterium, including a solution ofbacteria suspended therein, which was prepared by smearing a growthmedium, such as blood agar medium, normal agar medium, heart infusionagar medium, brain heart infusion agar medium, soybean/casein/digestagar medium, chocolate agar medium, egg-yolk-containing mannitol saltagar medium, or MRSA selection medium, with an analyte substance, suchas urine, pus, spinal fluid, secreted material, or puncture fluidobtained from a suspected patient and culturing the same under aerobicconditions at 35° C. to 37° C. for 18 hours or longer, and a culturesolution resulting from shake culture in liquid culture medium, bloodculture medium, or the like under aerobic conditions at 35° C. to 37° C.for 18 hours or longer. The term also refers to an extract deduced tocontain PBP2′ released from the cell wall due to pretreatment of such asuspension of bacteria. The “bacterium producing acell-wall-synthesizing enzyme, PBP2′” includes multidrug-resistantstaphylococcus, and the “multidrug-resistant staphylococcus” includesmultidrug-resistant Staphylococcus aureus (MRSA) and multidrug-resistantcoagulase-negative staphylococci (MRCNS). A solution of an analytesubstance, such as a urine, pus, spinal fluid, secreted material,puncture fluid, or another sample obtained from a suspected patientsuspended directly in physiological saline or phosphate buffer can alsobe a sample. From the viewpoint of detection sensitivity, use of asolution containing bacteria cultured in the above-described manner ispreferable.

The immunochromatography detection device of the present invention is animmunochromatography test piece. For example, such device is composed asshown in FIG. 1. Such device comprises on sheet-like solid-phasesupport; a sample supply site 1 to which a sample is supplied; a labeledreagent site 2 that holds a reagent, which is a labeled antibody bindingspecifically to PBP2′, in a manner such that the reagent is able tospread across the solid-phase support; and a capture reagent site 3 onwhich a capture reagent capable of specifically binding to and capturinga complex of PBP2′ and the labeled reagent has been immobilized. When asample is supplied to the sample supply site 1, the sample passesthrough the a labeled reagent site 2 and the capture reagent site 3 inthat order. In the present invention, a mixture of a sample and areagent that is a labeled antibody binding specifically to PBP2′ may besupplied to the sample supply site 1. In such a case, the labeledreagent site 2 on the solid-phase support may be omitted (FIG. 2). Whenthe mixture of a sample and a reagent that is a labeled antibody bindingspecifically to PBP2′ is to be supplied to the sample supply site 1,PBP2′ contained in the sample comes into contact with the labeledreagent at a site separated from a solid-phase support in advance. Thephrase “an analyte comes into contact with a labeled reagent at a siteseparated from a solid-phase support in advance” refers to conditionssuch that the labeled reagent is not contained on the solid-phasesupport, or it is in contact with the solid-phase support, and such thata liquid is not included at a site that can communicate with thesolid-phase support, such as a sample supply site, of theimmunochromatography detection device. In such a case, an analyte comesinto contact with the labeled reagent in advance, apart from thesolid-phase support or a site that is in contact with the solid-phasesupport.

The immunochromatography detection device of the present invention mayfurther comprise a control reagent and an absorption site. A controlreagent is not particularly limited. For example, a substance to whichan antibody in a labeled reagent binds may be used. A control reagentmay be immobilized at a site downstream of the capture reagent site. InFIG. 1, a control site 4 corresponds to such site. An absorption site iscapable of absorbing a liquid such that it absorbs a sample that haspassed through the capture site to regulate the flow of the sample. Suchsite may be provided at the lowermost site of the detection device. InFIG. 1, an absorption site 6 corresponds to such site. For example, anabsorption site made of paper may be used as an absorbent pad.

In the immunochromatography detection device of the present invention, asample supply site may be constituted by an end of a solid-phase supportas is, or it can be constituted by a member different from a solid-phasesupport. In the case of the latter constitution, a sample supply site isprovided in contact with a solid-phase support, so that a solution canspread and migrate to the solid-phase support with the aid of capillaryflow, in order that a sample supply site first absorbs a sample, or amixture of a sample and a labeled reagent, and then supplies theabsorbed sample or mixture to the solid-phase support. Examples ofmembers other than a solid-phase support include, but are not limitedto, members composed of natural or synthetic polymers of nitrocellulose,cellulose acetate, nylon, polyethersulfone, polyvinyl alcohol,polyester, glass fiber, polyolefin, cellulose, or polystyrene and amixture of such substances.

The “labeled reagent” of the immunochromatography detection device ofthe present invention is a conjugate of an antibody binding specificallyto PBP2′ and an adequate label substance. Examples of a label substanceinclude metal colloids, such as gold colloid, nonmetal colloids, such asselenium colloid, and insoluble substances such as colored resinparticles. In the present invention, such label substances areoccasionally referred to as “insoluble carriers.” Preferably, insolublecarriers are negatively charged. In general, a labeled reagent isimpregnated with a member different from a solid-phase support, dried,and then placed at a site continuous with the solid-phase support.Alternatively, the solid-phase support may be directly applied with thelabeled reagent and then dried. When the sample reaches a labeledreagent site containing a labeled reagent, the labeled reagent isdissolved in the sample, and the resulting solution can then be spreadacross the solid-phase support. Specifically, a labeled reagent is heldat a labeled reagent site in a manner extensible on the support.

The capture reagent of the immunochromatography detection device of thepresent invention is an antibody binding specifically to PBP2′, acapture reagent site can specifically bind to and capture a complex ofPBP2′ and a labeled reagent, and a complex of a labeled reagent/PBP2′/acapture reagent is then formed. In general, a capture reagent isprepared via direct applying of a solid-phase support, followed bydrying, although the method of preparation is not limited thereto. Amember other than a solid-phase support may be impregnated with acapture reagent and then dried, and the resultant may be placed on thesolid-phase support. A method for immobilizing a capture reagent on asolid-phase support is not limited to adsorption. Immobilization may becarried out by a conventional technique, such as chemical binding withthe use of a functional group such as an amino or carboxyl group.

An antibody to be used as a capture reagent may be same as an antibodyto be used as a labeled reagent. When there is only one site that bindsto such substance in PBP2′, however, a complex of a labeledreagent/PBP2′/a capture reagent would not be formed. In such a case,accordingly, a capture reagent is required to bind to a site of PBP2′that is different from a site to which a labeled reagents binds.

A solid-phase support may be of any substance, provided that a samplecan be absorbed by such support and fluidized via capillary phenomenon.For example, a support is selected from the group consisting of anatural or synthetic polymer of nitrocellulose, cellulose acetate,nylon, polyethersulfone, polyvinyl alcohol, polyester, glass fiber,polyolefin, cellulose, or polystyrene and a mixture of such substances.A solid-phase support is preferably strip-shaped.

The cell-wall-synthesizing enzyme, PBP2′, that is produced specificallyby a bacterium of multidrug-resistant staphylococcus is present on acell membrane located inside the cell wall. Pretreatment of breaking ormelting the cell wall of multidrug-resistant staphylococcus facilitatesa capture reagent and a labeled reagent to recognize PBP2′. As anextraction reagent for breaking or melting the cell wall ofmultidrug-resistant staphylococcus, a cell-wall-digesting enzyme or agiven surfactant having bactericidal action, such as a given cationicsurfactant or ampholytic surfactant, is known. Use of such reagents isdifficult because such reagents are expensive and strongly inhibit anantigen-antibody reaction at the time of immunochromatography detection,and for other reasons. Thus, use of a dilute alkaline solution ispreferable as an extraction reagent for PBP2′ at the time ofimmunochromatography detection in the present invention. Specificexamples thereof include an aqueous solution of 0.01N to 1.0N alkalimetal hydroxide or carbonate and an aqueous solution of 0.01N to 1.0Nalkaline earth metal hydroxide or carbonate. The pH of such dilutealkaline solution is preferably 11 or higher.

When a solution containing bacteria is pretreated with an dilutealkaline solution as described above, an antigen-antibody reaction wouldbe inhibited if a pH remains at 11 or higher after the pretreatment.Thus, neutralization of an alkaline solution is required. Aneutralization solution is not particularly limited, and examplesthereof include phosphate buffer, Tris buffer, and Good's buffer, suchas MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO,TAPSO, POPSO, HEPPSO, EPPS, Tricine, Bicine, or TAPS, exhibitingbuffering capacity at the optimal pH of the reaction system, i.e.,between 6 and 8, for immunochromatography detection. In the presentinvention, pretreatment of bacteria is a process of extracting abacterium producing a cell-wall-synthesizing enzyme, PBP2′, and moreparticularly, PBP2′ as an antigen by exposing a solution of bacteriadeduced to contain multidrug-resistant staphylococcus to alkalineconditions. “Pretreatment” also refers to a process of neutralizingalkaline conditions. Pretreatment may be carried out by adding analkaline solution to a sample solution comprising bacteria foralkalinization and then adding a neutralizing solution thereto.Alternatively, bacteria collected with the use of a platinum loop orcotton swab may be suspended directly in an alkaline solution, and aneutralizing solution may then be added. The amounts of such solutionsto be added are not limited, and such amounts may be determined so as tobring the pH of the neutralized sample solution to around 6 to 9. Also,a sample supply site may be impregnated with a neutralizing solution inadvance and then dried.

In the present invention, the solution of bacteria may be pretreated andthen neutralized, additives, such as salt, a surfactant, a protein, apolymer, an acidic compound, and a basic compound, may be added thereto,the resultant may be thoroughly mixed, and the sample may then besupplied to the sample supply site of the immunochromatography detectiondevice. These additives can enhance sensitivity of an antigen-antibodyreaction and can reduce false-positive reactions (e.g., false-positivereactions due to nonspecific reactions). Further, such additives may beadded to the aforementioned dilute alkaline solution or neutralizingsolution in advance to reduce procedural steps.

When detection of multidrug-resistant staphylococcus is intended,substances derived from non-multidrug-resistant staphylococci (MSSA orMSCNS) or bacteria other than staphylococci may occasionally bind toantibodies (i.e., as a labeled reagent and a capture reagent) and mayshow false-positive results. Thus, with the use of a PBP2′ detectionreagent used for latex agglutination assay, false-positive causativeagents, such as protein A, that are present on the cell wall ofStaphylococcus aureus exhibiting the capacity for binding to IgG had tobe inactivated by boiling a solution of bacteria in the presence of analkaline reagent, remnants after cell fracturing after centrifugationhad to be eliminated, and the supernatant after centrifugation had to beused for testing. Via immunochromatography detection according to thepresent invention, a capture reagent site of the immunochromatographydetection device may be impregnated with a surfactant to inhibit bindingof a false-positive causative agent to a capture antibody, withoutinactivating the false-positive causative agent by boiling. Anampholytic surfactant, an anionic surfactant, or a nonionic surfactantis preferable, and including one of them may be sufficient. Inhibitionof binding of a false-positive causative agent to a capture antibodywith the aid of a surfactant is considered to result from masking of asite of an antibody to which a false-positive causative agent binds by asurfactant. Accordingly, surfactants with larger molecular weightsand/or greater numbers of cyclic structures are considered to be moreeffective. When any of the following nonionic surfactants are used, forexample, false-positive results originating from non-multidrug-resistantStaphylococcus aureus that is not multidrug-resistant are more inhibitedcompared with the case where no surfactant was added: a nonionicsurfactant having a molecular weight of 646 and a single 6-membered ringstructure (tradename: Tx100; Nacalai Tesque); an ampholytic surfactanthaving a molecular weight of 364 without a 6-membered ring structure(tradename: SB3-14; Calbiochem); an ampholytic surfactant having amolecular weight of 615, three 6-membered ring structures, and a single5-membered ring structure (tradename: CHAPS; DOJINDO LABORATORIES); or anonionic surfactant having a molecular weight of 878, and three6-membered ring structures, and a single 5-membered ring structure(tradename: BIGCHAP, DOJINDO LABORATORIES). The effects of inhibitionattained with the use of CHAPS and BIGCHAP were greater than thoseattained with the use of Tx100 and SB3-14. Accordingly, the molecularweight of a surfactant is considered to be preferably 300 or higher, andmore preferably 600 or higher, and a surfactant is considered topreferably comprise a greater number of cyclic structures, such as6-membered rings. For the purpose of inhibiting false-positivereactions, use of a surfactant that is charged in the same manner aswith the case of an insoluble carrier is preferable. When an insolublecarrier is a negatively charged latex or gold colloid, it is preferablethat a surfactant also be negatively charged.

A false-positive reaction resulting from protein A present in the cellwall of Staphylococcus aureus is of particularly serious concern.Protein A strongly binds to the Fc region of immunoglobulin G.Antibodies that specifically recognize PBP2′ are used as labeledreagents and capture reagents. Antibodies may be immunoglobulin G orimmunoglobulin M, without particular limitation. When immunoglobulin Gis used, such antibody is used while eliminating the Fc region, and acapture reagent region is combined with the aforementioned surfactant.Thus, a false-positive reaction caused by protein A can be avoided. TheFc region can be easily eliminated with the use of a known degradingenzyme, such as pepsin or papain.

Protein G contained in the cell wall of group G Streptococcus also bindsspecifically to the Fc region of immunoglobulin G. When a samplesolution contains group G Streptococcus, accordingly, protein G maynonspecifically react with a labeled reagent or a capture reagent, aswith the case of protein A. Such false-positive reaction can also beavoided in the above-described manner.

Among surfactants to be applied to the capture reagent site, propertiesof an ionic surfactant are particularly affected by the concentration ofanions or cations in the solution that passes through the capturereagent region. This necessitates determination of the optimalconcentration of anions or cations in the solution in accordance withthe type, concentration, properties, or other conditions of a surfactantto be used. Examples of anions include a chloride ion, a bromide ion,and an iodide ion having a high ionization tendency. Examples of cationsinclude a potassium ion, a calcium ion, a sodium ion, and a magnesiumion having a high ionization tendency. In this case, a plurality ofanions or cations may be used.

Detection sensitivity can be improved by adding a surfactant containinga sulfobetaine region to a capture reagent site, which is the methodaccording to the present invention. Since PBP2′ could not be detectedwith high sensitivity with the use of a PBP2′ detection reagent vialatex agglutination assay, the cell wall had to be broken via boiling ofa solution of bacteria in the presence of an alkaline reagent in orderto completely extract PBP2′. According to the present invention,detection sensitivity is improved with the addition of a surfactantcontaining a sulfobetaine region, which enabled detection of PBP2′ withhigh sensitivity even when elution of PBP2′ is incomplete in the absenceof boiling. Boiling in the presence of an alkaline reagent would causePBP2′ degradation, or would cause desensitization via denaturation of anantibody recognition site due to structural changes; however, detectionsensitivity can be advantageously maintained with the absence ofboiling.

Examples of surfactants each containing a sulfobetaine region includeCHAPS, CHAPSO, myristylsulfobetaine (SB3-14), and dodecyl dimethylammonio butane sulfonate (DDABS).

A surfactant for inhibiting binding of the false-positive causativeagent to a capture antibody, and a surfactant for improving sensitivityare added to a capture reagent in advance when a capture reagent is tobe immobilized at a capture reagent site. Either or both of a surfactantfor inhibiting binding of the false-positive causative agent to acapture antibody and a sulfobetaine-type surfactant capable of improvingsensitivity may be used. Alternatively, a surfactant for inhibitingbinding of the false-positive causative agent to a capture antibody maybe a sulfobetaine-type surfactant, and sensitivity can be improved insuch a case.

Further, the method of the present invention can produce the effect ofinhibiting binding of a false-positive causative agent to a captureantibody and the effect of improving sensitivity by adding a surfactantcontaining a sulfobetaine region having a molecular weight of preferably300 or higher, and more preferably 600 or higher, to a capture reagentsite. Specifically, CHAPS and SB3-14 are preferable, with CHAPS beingparticularly preferable. A plurality of surfactants may be added to thecapture reagent site.

When an anion or cation is added as described above, such anion orcation may be added to a sample solution deduced to containmultidrug-resistant staphylococcus or a sample solution deduced tocontain PBP2′ released from the cell wall via sample pretreatment.

The method of the present invention further includes a method ofpreventing a false-positive causative agent from reaching the capturereagent site, thereby preventing influences of such false-positivecausative agent. For example, a false-positive causative agent may beeliminated at a site upstream of the capture reagent site in the deviceof the present invention. As a means for eliminating a false-positivecausative agent, a sample supply site of the immunochromatographydetection device preferably comprises glass fibers. A bacteria-derivedfalse-positive causative agent is adsorbed by glass fibers, whichinhibits the false-positive causative agent from passing through thecapture reagent. This can consequently inhibit a false-positivereaction.

The method of the present invention is a detection method based on anantigen-antibody reaction using a cell-wall-synthesizing enzyme, PBP2′,as an antigen and an antibody reacting therewith. If acell-wall-synthesizing enzyme, PBP2′, is present in the sample,accordingly, PBP2′ can be detected by the method of the presentinvention. In general, a cell-wall-synthesizing enzyme, PBP2′, isproduced specifically by a multidrug-resistant staphylococcus, and ispresent specifically in such bacterium.

At present, the existence of PBP2′-producing bacteria other thanmultidrug-resistant staphylococcus is not known. In the presentdescription, the term “multidrug-resistant staphylococcus” isoccasionally used as a specific example instead of the term “a bacteriumproducing a cell-wall-synthesizing enzyme, PBP2′.” Such use is notintended to limit the bacterium to “a multidrug-resistantstaphylococcus.” This term can be read as “a bacterium producing acell-wall-synthesizing enzyme, PBP2′” within the scope that allows themethod of the present invention to be implemented. That is, bacteriaproducing PBP2′ other than multidrug-resistant staphylococcus can bedetected by the method of the present invention. Further, bacteria eachcontaining a cell-wall-synthesizing enzyme, PBP2′, can be detected bythe method of the present invention. In general, acell-wall-synthesizing enzyme, PBP2′, is considered to be presentspecifically on a cell membrane inside the cell wall of amultidrug-resistant staphylococcus. The method of the present inventioninvolves the use of a PBP2′ extraction reagent used for pretreatment ofbreaking or melting the cell membrane. Accordingly, bacteria eachcomprising a cell-wall-synthesizing enzyme, PBP2′, inside the cell wallcan be effectively detected by the method of the present invention.

EXAMPLES

Hereafter, the present invention is described in greater detail withreference to the following examples, although the present invention isnot limited thereto.

Example 1 Detection of PBP2′ in MRSA Via Immunochromatography Detection(FIG. 1)

(1) Preparation and Drying of Latex Particles Sensitized with Antibody

An anti-PBP2′ monoclonal antibody was treated with pepsin in accordancewith conventional technique to obtain F(ab′)2. The resultant wassubjected to sensitization of 0.4 μm of latex particles, and theresulting solution was sprayed on an unwoven polystyrene fabric. Theresultant was then dried under reduced pressure for 1 hour in adecompression device to prepare a dry latex antibody pad. The pad wascut at intervals of 4 mm for use and used as a labeled site 2.

(2) Preparation of Immunochromatography Detection Device

A second anti-PBP2′ monoclonal antibody having a recognition sitedifferent from that of the anti-PBP2′ monoclonal antibody used for latexsensitization was treated with pepsin in accordance with a conventionaltechnique to obtain F(ab′)2. The resultant was diluted with a citratebuffer (pH 6) containing 0.075% CHAPS, the resultant was applied to anitrocellulose membrane (solid-phase support 5), and the membrane wasthoroughly dried (capture reagent site 3). As a control reagent,anti-mouse IgGs was applied to a nitrocellulose membrane in the samemanner and thoroughly dried (control site 4).

A capture reagent site 3 and a solid-phase support 5 including a controlsite 4 were provided on a hydrophobic sheet 7, and a labeled reagentsite 2, a glass fiber as a sample supply site 1, and a filter paper asan absorption site 6 were provided at any positions.

(3) Pretreatment of Sample

Staphylococcus aureus (14 bacteria) were cultured on blood agar mediumat 35° C. overnight, and a loopful of culture was suspended directly in0.2N NaOH. Similarly, a colony was picked up and then suspended in 100μl of 0.2N NaOH. The resultant was then neutralized with 50 μl of 0.6MTris-HCl buffer containing a nonionic surfactant, bovine serum albumin,and rabbit IgG.

(4) Assay

The immunochromatography detection device was introduced into a 1.5-mltube containing a solution after pretreatment of samples, colordevelopment of a capture reagent 3 was evaluated with the naked eye tenminutes later, and the capture reagent that had developed color wasidentified as positive. The reagent that had not developed color wasidentified as negative.

(5) Drug Sensitivity Test

Bacteria cultured in the same manner as in (3) were suspended in asterilized 0.9% sodium chloride solution, and the absorbance at 578 nmof the solution was adjusted to 0.3. The resulting solution was sowed onMuller-Hinton agar medium containing 6 μg/ml oxacillin and 4% sodiumchloride, culture was conducted at 35° C. for 24 hours, the bacteriathat had grown were determined to be MRSA, and the bacteria that had notgrown were determined to be MSSA.

(6) Results

The results are shown in Table 1.

TABLE 1 Drug sensitivity MRSA MSSA Total One loopful Present MRSA 4 0 4invention MSSA 0 10 10 Total 4 10 14 One colony Present MRSA 4 0 4invention MSSA 0 10 10 Total 4 10 14

As is apparent from Table 1, the positive concordance rate between MRSAand MSSA is 100% (4/4), and the negative concordance rate therebetweenis 100% (10/10). This indicates that the concordance rate as a whole is100% (14/14). These results represent the superiority of presentinvention to conventional techniques.

Example 2 Effect of Addition of Surfactant to Capture Reagent Site

(1) Preparation and Drying of Latex Particles Sensitized with Antibody

The latex particles prepared in Example 1 were used.

(2) Preparation of Immunochromatography Detection Device

The device was prepared in the same manner as in Example 1. In thiscase, an device comprising a capture reagent site and 0.05% CHAPS,Tx100, SB3-14, BIGCHAP, and DTAC (dodecyl trimethyl ammonium chloride)added thereto and an device comprising no such substances were preparedand used.

(3) Pretreatment of Sample

A bacterium that had been evaluated as being drug-sensitive, i.e., MSSA,and a bacterium that had been evaluated as being drug-resistant, i.e.,MRSA, via the drug sensitivity test were cultured in the same manner asin Example 1. Two and three loopfuls of MSSA and a loopful of MRSA werepicked up and then pretreated.

(4) Assay

An MSSA was tested in the same manner as in Example 1 without dilution.Further, the MRSA was subjected to two-step dilution with a mixturecomprising 0.2N NaOH and 0.6M Tris HCl buffer (containing a nonionicsurfactant, bovine serum albumin, and rabbit IgG) at 2:1, and 150 μlthereof diluted at least 1024-fold was tested in the same manner as inExample 1.

(5) Results

The results attained with the device comprising a surfactant added to acapture reagent site and the results attained with the device comprisingno surfactant were compared. The results attained with the use of MSSAare shown in Table 2. The negative results are represented by “−,” andthe degree of false-positive reaction are represented by the number of“+”'s. The results attained with the use of MRSA are shown in Table 3. Anegative result is represented by “−,” and a positive result isrepresented by “+.”

TABLE 2 MSSA Without addition Tx 100 CHAPS SB3-14 BIGCHAP DTAC MSSA (2loopfuls) + − − − − + MSSA (3 loopfuls) ++ + − + − ++

When a surfactant was not added and a cationic surfactant, DTAC, wasadded, a false-positive result was attained with 2 loopfuls of MSSA.When Tx 100 or SB3-14 having a relatively small molecular weight wasadded, 3 loopfuls thereof caused a false-positive reaction, although thedegree thereof was not as strong as that attained when a surfactant wasnot added or when DTAC was added. When CHAPS or BIGCHAP having arelatively large molecular weight was added, 2 and 3 loopfuls thereofyielded a negative result.

As is apparent from Table 2, addition of a nonionic surfactant, i.e.,Tx100 or BIGCHAP, or an ampholytic surfactant, i.e., CHAPS or SB3-14, toa capture reagent site can inhibit a false-positive reaction originatingfrom MSSA. Also, CHAPS or BIGCHAP comprising a large number of cyclicstructures and having a large molecular weight can more effectivelyinhibit a false-positive reaction. These results demonstrate thesuperiority of the addition of a surfactant to a capture reagent site.

TABLE 3 MRSA Without addition Tx 100 CHAPS SB3-14 BIGCHAP DTAC1:1024 + + + + + + (false-positive) 1:2048 − − + + − + (false-positive)1:4096 − − − − − + (false-positive)

Detection sensitivity was improved with the addition of CHAPS or SB3-14to a capture reagent site. The results attained with the addition ofDTAC were considered to constitute a false-positive.

As is apparent from Table 3, the addition of a sulfobetaine-typesurfactant, i.e., SB3-14 or CHAPS, to a capture reagent site can improvesensitivity, compared with cases in which no surfactant was added or anon-sulfobetaine-type surfactant, i.e., Tx100 or BIGCHAP, was added.These results demonstrate superiority of the addition of asulfobetaine-type surfactant to a capture reagent site.

The results of Example 2 demonstrate that CHAPS particularly showssatisfactory effects of inhibiting a false-positive reaction and thoseof improving sensitivity and that CHAPS is thus particularly useful.

Example 3 Detection of PBP2′ in MRCNS Via Immunochromatography Detection

(1) Preparation and Drying of Latex Particles Sensitized with Antibody

The latex particles prepared in Example 1 were used.

(2) Preparation of Immunochromatography Detection Device

The device was prepared in the same manner as in Example 1.

(3) Pretreatment of Sample

Among staphylococci that exhibit properties of staphylococci via gramstaining and that had been evaluated to be positive via a catalase testand to be negative via a coagulase test, two bacteria that had beenevaluated as being methicillin-sensitive, i.e., MSCNS, via a drugsensitivity test and a bacterium that had been evaluated as beingmethicillin-resistant, i.e., MRCNS, via a drug sensitivity test werecultured in the same manner as in Example 1, and a loopful thereof waspicked and then subjected to pretreatment.

(4) Assay

Assay was conducted in the same manner as in Example 1.

(5) Drug Sensitivity Test

Test was conducted in the same manner as in Example 1.

(6) Results

The results are shown in Table 4. The results of the drug sensitivitytest are represented as follows: S: sensitivity; R: resistance; +:positive result; and −: negative result.

TABLE 4 Drug sensitivity Present test invention Strain A S − Strain B S− Strain C R +

The two strains that had been evaluated as being sensitive, i.e., MSCNS,via the drug sensitivity test showed negative results, and a strain thathad been evaluated as being resistant, i.e., MRCNS, via the drugsensitivity test showed positive results.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

1. A method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, comprising detection of acell-wall-synthesizing enzyme, PBP2′, via immunochromatography detectionbased on an antigen-antibody reaction.
 2. The method for detecting abacterium that produces a cell-wall-synthesizing enzyme, PBP2′,according to claim 1, which involves the use of an immunochromatographydetection device comprising on a sheet-like solid-phase support: asample supply site to which a sample solution deduced to contain abacterium producing a cell-wall-synthesizing enzyme, PBP2′ or a solutiondeduced to contain PBP2′ released from the cell wall via samplepretreatment is supplied; a labeled reagent site that holds a reagent,which is a labeled antibody binding specifically to PBP2′, in a mannersuch that the reagent is able to spread across the solid-phase support;and a capture reagent site on which a capture reagent capable ofspecifically binding to and capturing a complex of PBP2′ and the labeledreagent has been immobilized.
 3. The method for detecting a bacteriumthat produces a cell-wall-synthesizing enzyme, PBP2′, according to claim1, wherein PBP2′ comes into contact with the labeled reagent at a siteseparated from a solid-phase support in advance, and a sample-reagentmixture comprising a sample solution deduced to contain a bacteriumproducing a cell-wall-synthesizing enzyme, PBP2′ or a solution deducedto contain PBP2′ released from the cell wall via sample pretreatment andthe labeled reagent that is a labeled antibody binding specifically toPBP2′ is supplied to the sample supply site.
 4. The method for detectinga bacterium that produces a cell-wall-synthesizing enzyme, PBP2′,according to claim 2, wherein the labeled reagent is an insolublecarrier to which an antibody is bound.
 5. The method for detecting abacterium that produces a cell-wall-synthesizing enzyme, PBP2′,according to claim 2, wherein the capture reagent site comprises anampholytic surfactant, an anionic surfactant, and/or a nonionicsurfactant.
 6. The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to claim 2, wherein thecapture reagent site comprises a sulfobetaine-type surfactant.
 7. Themethod for detecting a bacterium that produces a cell-wall-synthesizingenzyme, PBP2′, according to claim 5, wherein an anion having a highionization tendency is added to a sample solution deduced to contain abacterium producing a cell-wall-synthesizing enzyme, PBP2′, or asolution deduced to contain PBP2′ released from the cell wall via samplepretreatment prior to supply of such solution to a sample supply site.8. The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to claim 7, wherein theanion having a high ionization tendency is at least one anion selectedfrom the group consisting of a chloride ion, a bromide ion, and aniodide ion.
 9. The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to claim 5, wherein acation having a high ionization tendency is added to a sample solutiondeduced to contain a bacterium producing a cell-wall-synthesizingenzyme, PBP2′, or a solution deduced to contain PBP2′ released from thecell wall via sample pretreatment prior to supply of such solution to asample supply site.
 10. The method for detecting a bacterium thatproduces a cell-wall-synthesizing enzyme, PBP2′, according to claim 9,wherein the cation having a high ionization tendency is at least onecation selected from the group consisting of a potassium ion, a calciumion, a sodium ion, and a magnesium ion.
 11. The method for detecting abacterium that produces a cell-wall-synthesizing enzyme, PBP2′,according to claim 1, which comprises a step of pretreating a sample viaalkaline treatment or neutralization.
 12. The method for detecting abacterium that produces a cell-wall-synthesizing enzyme, PBP2′,according to claim 11, wherein the alkaline treatment is carried outusing an aqueous solution of alkali metal hydroxide or carbonate or anaqueous solution of alkaline earth metal hydroxide or carbonate.
 13. Themethod for detecting a bacterium that produces a cell-wall-synthesizingenzyme, PBP2′, according to claim 12, wherein the pH of the aqueoussolution of alkali metal hydroxide or carbonate or the aqueous solutionof alkaline earth metal hydroxide or carbonate is 11 or higher.
 14. Themethod for detecting a bacterium that produces a cell-wall-synthesizingenzyme, PBP2′, according to claim 12, wherein the concentration of thealkali metal hydroxide or carbonate or alkaline earth metal hydroxide orcarbonate is between 0.01 N and 1.0N.
 15. The method for detecting abacterium that produces a cell-wall-synthesizing enzyme, PBP2′,according to claim 11, wherein the aqueous solution after alkalitreatment is neutralized with a buffer.
 16. The method for detecting abacterium that produces a cell-wall-synthesizing enzyme, PBP2′,according to claim 1, wherein the sample supply site comprises glassfibers.
 17. The method for detecting a bacterium that produces acell-wall-synthesizing enzyme, PBP2′, according to claim 1, wherein thebacterium producing a cell-wall-synthesizing enzyme, PBP2′, is amultidrug-resistant staphylococcus.
 18. An immunochromatographydetection device for detecting a bacterium producing a cell wallsynthesizing enzyme, PBP2′, comprising on a sheet-like solid-phasesupport: a sample supply site to which a sample solution deduced tocontain a bacterium producing a cell-wall-synthesizing enzyme, PBP2′ ora solution deduced to contain PBP2′ released from the cell wall viasample pretreatment is supplied; a labeled reagent site that holds areagent, which is a labeled antibody binding specifically to PBP2′, in amanner such that the reagent is able to spread across the solid-phasesupport; and a capture reagent site on which a capture reagent capableof specifically binding to and capturing a complex of PBP2′ and thelabeled reagent has been immobilized, the capture reagent sitecomprising an ampholytic surfactant, an anionic surfactant, and/or anonionic surfactant.
 19. The immunochromatography detection device fordetecting a bacterium producing a cell wall synthesizing enzyme, PBP2′,according to claim 18, wherein the capture reagent site comprises asulfobetaine-type surfactant.
 20. The immunochromatography detectiondevice for detecting a bacterium producing a cell wall synthesizingenzyme, PBP2′, according to claim 18, wherein the sample supply sitecomprises glass fibers.
 21. The immunochromatography detection devicefor detecting a bacterium producing a cell wall synthesizing enzyme,PBP2′, according to claim 18, wherein the bacterium producing acell-wall-synthesizing enzyme, PBP2′, is a multidrug-resistantstaphylococcus.
 22. A kit for detecting a bacterium producing acell-wall-synthesizing enzyme, PBP2′, comprising: theimmunochromatography detection device for detecting a bacteriumproducing a cell wall synthesizing enzyme, PBP2′, according to claim 18;and an anion or cation solution having a high ionization tendency to beadded to a sample solution deduced to contain a bacterium producing acell-wall-synthesizing enzyme, PBP2′, or a solution deduced to containPBP2′ released from the cell wall via sample pretreatment.
 23. The kitfor detecting a bacterium producing a cell-wall-synthesizing enzyme,PBP2′, according to claim 22, wherein the anion having a high ionizationtendency is at least one anion selected from the group consisting of achloride ion, a bromide ion, and an iodide ion.
 24. The kit fordetecting a bacterium producing a cell-wall-synthesizing enzyme, PBP2′,according to claim 22, wherein the cation having a high ionizationtendency is at least one cation selected from the group consisting of apotassium ion, a calcium ion, a sodium ion, and a magnesium ion.
 25. Thekit for detecting a bacterium producing a cell-wall-synthesizing enzyme,PBP2′, according to claim 22, which further comprises an alkali solutionfor alkali treatment of a sample with alkali and a buffer forneutralization.
 26. The kit for detecting a bacterium producing acell-wall-synthesizing enzyme, PBP2′, according to claim 25, wherein thealkali solution is an aqueous solution of alkali metal hydroxide orcarbonate or an aqueous solution of alkaline earth metal hydroxide orcarbonate.
 27. The kit for detecting a bacterium producing acell-wall-synthesizing enzyme, PBP2′, according to claim 26, wherein thepH of the aqueous solution of alkali metal hydroxide or carbonate or theaqueous solution of alkaline earth metal hydroxide or carbonate is 11 orhigher.
 28. The kit for detecting a bacterium producing acell-wall-synthesizing enzyme, PBP2′, according to claim 26, wherein theconcentration of the alkali metal hydroxide or carbonate or alkalineearth metal hydroxide or carbonate is between 0.01N and 1.0N.
 29. Thekit for detecting a bacterium producing a cell-wall-synthesizing enzyme,PBP2′, according to claim 22, wherein the bacterium producing acell-wall-synthesizing enzyme, PBP2′, is a multidrug-resistantstaphylococcus.